The apocarotenoid ß-ionone regulates the transcriptome of Arabidopsis thaliana and increases its resistance against Botrytis cinerea.

Carotenoids are isoprenoid pigments indispensable for photosynthesis. Moreover, they are the precursor of apocarotenoids, which include the phytohormones abscisic acid (ABA) and strigolactones (SLs) as well as retrograde signaling molecules and growth regulators, such as ß-cyclocitral and zaxinone. Here, we show that the application of the volatile apocarotenoid ß-ionone (ß-I) to Arabidopsis plants at micromolar concentrations caused a global reprogramming of gene expression, affecting thousands of transcripts involved in stress tolerance, growth, hormone metabolism, pathogen defense, and photosynthesis. This transcriptional reprogramming changes, along with induced changes in the level of the phytohormones ABA, jasmonic acid, and salicylic acid, led to enhanced Arabidopsis resistance to the widespread necrotrophic fungus Botrytis cinerea (B.c.) that causes the gray mold disease in many crop species and spoilage of harvested fruits. Pre-treatment of tobacco and tomato plants with ß-I followed by inoculation with B.c. confirmed the effect of ß-I in increasing the resistance to this pathogen in crop plants. Moreover, we observed reduced susceptibility to B.c. in fruits of transgenic tomato plants overexpressing LYCOPENE ß-CYCLASE, which contains elevated levels of endogenous ß-I, providing a further evidence for its effect on B.c. infestation. Our work unraveled ß-I as a further carotenoid-derived regulatory metabolite and indicates the possibility of establishing this natural volatile as an environmentally friendly bio-fungicide to control B.c.

Abscisic acid inhibits germination of Striga seeds and is released by them likely as a rhizospheric signal supporting host infestation.

Seeds of the root parasitic plant Striga hermonthica undergo a conditioning process under humid and warm environments before germinating in response to host-released stimulants, particularly strigolactones (SLs). The plant hormone abscisic acid (ABA) regulates different growth and developmental processes, and stress response; however, its role during Striga seed germination and early interactions with host plants is under-investigated. Here, we show that ABA inhibited Striga seed germination and that hindering its biosynthesis induced conditioning and germination in unconditioned seeds, which was significantly enhanced by treatment with the SL analog rac-GR24. However, the inhibitory effect of ABA remarkably decreased during conditioning, confirming the loss of sensitivity towards ABA in later developmental stages. ABA measurement showed a substantial reduction of its content during the early conditioning stage and a significant increase upon rac-GR24-triggered germination. We observed this increase also in released seed exudates, which was further confirmed by using the Arabidopsis ABA-reporter GUS marker line. Seed exudates of germinated seeds, containing elevated levels of ABA, impaired the germination of surrounding Striga seeds in vitro and promoted root growth of a rice host towards germinated Striga seeds. Application of ABA as a positive control caused similar effects, indicating its function in Striga/Striga and Striga/host communications. In summary, we show that ABA is an essential player during seed dormancy and germination processes in Striga and acts as a rhizospheric signal likely to support host infestation.

Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism.

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

The Arabidopsis D27-LIKE1 is a cis/cis/trans-ß-carotene isomerase that contributes to Strigolactone biosynthesis and negatively impacts ABA level.

The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-ß-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed an unreported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-ß-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the d27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed a more pronounced branching phenotype compared to d27. The contribution of AtD27-like1 to SL biosynthesis is likely a result of its formation of 9-cis-ß-carotene that was present at higher levels in AtD27-like1 overexpressing lines. By contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of ABA, in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing GUS driven by the AtD27LIKE1 promoter and transcript analysis of hormone-treated Arabidopsis seedlings revealed that AtD27LIKE1 is expressed in different tissues and affects ABA and auxin. Taken together, our work reports a cis/cis-ß-carotene isomerase that affects the content of both cis-carotenoid-derived plant hormones, ABA and SLs.

Protein interactome of 3',5'-cAMP reveals its role in regulating the actin cytoskeleton.

Identification of protein interactors is ideally suited for the functional characterization of small molecules. 3',5'-cAMP is an evolutionary ancient signaling metabolite largely uncharacterized in plants. To tap into the physiological roles of 3',5'-cAMP, we used a chemo-proteomics approach, thermal proteome profiling (TPP), for the unbiased identification of 3',5'-cAMP protein targets. TPP measures shifts in the protein thermal stability upon ligand binding. Comprehensive proteomics analysis yielded a list of 51 proteins significantly altered in their thermal stability upon incubation with 3',5'-cAMP. The list contained metabolic enzymes, ribosomal subunits, translation initiation factors, and proteins associated with the regulation of plant growth such as CELL DIVISION CYCLE 48. To functionally validate obtained results, we focused on the role of 3',5'-cAMP in regulating the actin cytoskeleton suggested by the presence of actin among the 51 identified proteins. 3',5'-cAMP supplementation affected actin organization by inducing actin-bundling. Consistent with these results, the increase in 3',5'-cAMP levels, obtained either by feeding or by chemical modulation of 3',5'-cAMP metabolism, was sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. The observed rescue was specific to 3',5'-cAMP, as demonstrated using a positional isomer 2',3'-cAMP, and true for the nanomolar 3',5'-cAMP concentrations reported for plant cells. In vitro characterization of the 3',5'-cAMP-actin pairing argues against a direct interaction between actin and 3',5'-cAMP. Alternative mechanisms by which 3',5'-cAMP would affect actin dynamics, such as by interfering with calcium signaling, are discussed. In summary, our work provides a specific resource, 3',5'-cAMP interactome, as well as functional insight into 3',5'-cAMP-mediated regulation in plants.

Towards a validated glossary of usability attributes for the evaluation of wearable robotic devices.

BACKGROUND: Despite technical advances in the field of wearable robotic devices (WRD), there is still limited user acceptance of these technologies. While usability often comes as a key factor influencing acceptance, there is a scattered landscape of definitions and scopes for the term. To advance usability evaluation, and to integrate usability features as design requirements during technology development, there is a need for benchmarks and shared terminology. These should be easily accessible and implementable by developers. METHODS: An initial set of usability attributes (UA) was extracted from a literature survey on usability evaluation in WRD. The initial set of attributes was enriched and locally validated with seven developers of WRD through an online survey and a focus group. The locally validated glossary was then externally validated through a globally distributed online survey. RESULTS: The result is the Robotics Usability Glossary (RUG), a comprehensive glossary of 41 UA validated by 70 WRD developers from 17 countries, ensuring its generalizability. 31 of the UA had high agreement scores among respondents and 27 were considered highly relevant in the field, but only 11 of them had been included as design criteria by the respondents. CONCLUSIONS: Multiple UA ought to be considered for a comprehensive usability assessment. Usability remains inadequately incorporated into device development, indicating a need for increased awareness and end-user perspective. The RUG can be readily accessed through an online platform, the Interactive Usability Toolbox (IUT), developed to provide context-specific outcome measures and usability evaluation methods. Overall, this effort is an important step towards improving and promoting usability evaluation practices within WRD. It has the potential to pave the way for establishing usability evaluation benchmarks that further endorse the acceptance of WRD.

2',3'-cAMP treatment mimics the stress molecular response in Arabidopsis thaliana.

The role of the RNA degradation product 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) is poorly understood. Recent studies have identified 2',3'-cAMP in plant material and determined its role in stress signaling. The level of 2',3'-cAMP increases upon wounding, in the dark, and under heat, and 2',3'-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2',3'-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2',3'-cAMP treatment. We demonstrated that 2',3'-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2',3'-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2',3'-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2',3'-cAMP treatment influences the movement of processing bodies, confirming the role of 2',3'-cAMP in the formation and motility of membraneless organelles.

Re-defining wearable robots: a multidisciplinary approach towards a unified terminology.

Effective communication is especially important in the wearable robots (WRs) community, which encloses a great variety of devices across different application domains, e.g., healthcare, occupational, and consumer. In this paper we present a vocabulary of terms with the aim to create a common understanding of terms and concepts among the different fields of expertise relevant in the WRs community. Our goal is to develop shared documentation that could serve as a reference to facilitate the use of accepted definitions in the field. The presented vocabulary is the result of different focus group discussions among experts in the field. The resulting document was then validated by presenting it to the WR community through an online survey. The results of the survey highlight a strong agreement in terms of acceptance of the vocabulary, its usefulness, and applicability of the proposed definitions as well as an overall appreciation for its purpose and target. This work represents a pilot study providing unique material for the WR community, encouraging the use of shared agreed definitions. The reported version of the vocabulary has been made available as a live document in a github repository, for public commenting and further improvements.

Review of electromyography onset detection methods for real-time control of robotic exoskeletons.

BACKGROUND: Electromyography (EMG) is a classical technique used to record electrical activity associated with muscle contraction and is widely applied in Biomechanics, Biomedical Engineering, Neuroscience and Rehabilitation Robotics. Determining muscle activation onset timing, which can be used to infer movement intention and trigger prostheses and robotic exoskeletons, is still a big challenge. The main goal of this paper was to perform a review of the state-of-the-art of EMG onset detection methods. Moreover, we compared the performance of the most commonly used methods on experimental EMG data. METHODS: A total of 156 papers published until March 2022 were included in the review. The papers were analyzed in terms of application domain, pre-processing method and EMG onset detection method. The three most commonly used methods [Single (ST), Double (DT) and Adaptive Threshold (AT)] were applied offline on experimental intramuscular and surface EMG signals obtained during contractions of ankle and knee joint muscles. RESULTS: Threshold-based methods are still the most commonly used to detect EMG onset. Compared to ST and AT, DT required more processing time and, therefore, increased onset timing detection, when applied on experimental data. The accuracy of these three methods was high (maximum error detection rate of 7.3%), demonstrating their ability to automatically detect the onset of muscle activity. Recently, other studies have tested different methods (especially Machine Learning based) to determine muscle activation onset offline, reporting promising results. CONCLUSIONS: This study organized and classified the existing EMG onset detection methods to create consensus towards a possible standardized method for EMG onset detection, which would also allow more reproducibility across studies. The three most commonly used methods (ST, DT and AT) proved to be accurate, while ST and AT were faster in terms of EMG onset detection time, especially when applied on intramuscular EMG data. These are important features towards movement intention identification, especially in real-time applications. Machine Learning methods have received increased attention as an alternative to detect muscle activation onset. However, although several methods have shown their capability offline, more research is required to address their full potential towards real-time applications, namely to infer movement intention.

A qualitative study to elicit user requirements for lower limb wearable exoskeletons for gait rehabilitation in spinal cord injury.

OBJECTIVE: We aim to determine a comprehensive set of requirements, perceptions, and expectations that people with spinal cord injury (SCI) and the clinicians in charge of their rehabilitation have regarding the use of wearable robots (WR) for gait rehabilitation. BACKGROUND: There are concerns due to the limited user acceptance of WR for gait rehabilitation. Developers need to emphasize understanding the needs and constraints of all stakeholders involved, including the real-life dynamics of rehabilitation centers. METHODS: 15 people with SCI, 9 without experience with WR and 6 with experience with these technologies, and 10 clinicians from 3 rehabilitation centers in Spain were interviewed. A directed content analysis approach was used. RESULTS: 78 codes grouped into 9 categories (physical results, usability, psychology-related codes, technical characteristics, activities, acquisition issues, context of use, development of the technologies and clinical rehabilitation context) were expressed by at least 20% of the users interviewed, of whom 16 were not found in the literature. The agreement percentage between each group and subgroup included in the study, calculated as the number of codes that more than 20% of both groups expressed, divided over the total amount of codes any of those two groups agreed on (≥ 20%), showed limited agreement between patients and clinicians (50.00%) and between both types of patients (55.77%). The limited accessibility and availability of lower limb exoskeletons for gait rehabilitation arose in most of the interviews. CONCLUSIONS: The limited agreement percentage between patients and clinicians indicates that including both types of users in the design process of these technologies is important, given that their requirements are complementary. Engaging users with prior technology experience is recommended, as they often exhibit strong internal consensus and articulate well-defined requirements. This study adds up the knowledge available in the literature and the new codes found in our data, which enlighten important aspects that ought to be addressed in the field to develop technologies that respond to users' needs, are usable and feasible to implement in their intended contexts. APPLICATION: The set of criteria summarized in our study will be useful to guide the design, development, and evaluation of WR for gait rehabilitation to meet user's needs and allow them to be implemented in their intended context of use.

Benchmarking the Effects on Human-Exoskeleton Interaction of Trajectory, Admittance and EMG-Triggered Exoskeleton Movement Control.

Nowadays, robotic technology for gait training is becoming a common tool in rehabilitation hospitals. However, its effectiveness is still controversial. Traditional control strategies do not adequately integrate human intention and interaction and little is known regarding the impact of exoskeleton control strategies on muscle coordination, physical effort, and user acceptance. In this article, we benchmarked three types of exoskeleton control strategies in a sample of seven healthy volunteers: trajectory assistance (TC), compliant assistance (AC), and compliant assistance with EMG-Onset stepping control (OC), which allows the user to decide when to take a step during the walking cycle. This exploratory study was conducted within the EUROBENCH project facility. Experimental procedures and data analysis were conducted following EUROBENCH's protocols. Specifically, exoskeleton kinematics, muscle activation, heart and breathing rates, skin conductance, as well as user-perceived effort were analyzed. Our results show that the OC controller showed robust performance in detecting stepping intention even using a corrupt EMG acquisition channel. The AC and OC controllers resulted in similar kinematic alterations compared to the TC controller. Muscle synergies remained similar to the synergies found in the literature, although some changes in muscle contribution were found, as well as an overall increase in agonist-antagonist co-contraction. The OC condition led to the decreased mean duration of activation of synergies. These differences were not reflected in the overall physiological impact of walking or subjective perception. We conclude that, although the AC and OC walking conditions allowed the users to modulate their walking pattern, the application of these two controllers did not translate into significant changes in the overall physiological cost of walking nor the perceived experience of use. Nonetheless, results suggest that both AC and OC controllers are potentially interesting approaches that can be explored as gait rehabilitation tools. Furthermore, the INTENTION project is, to our knowledge, the first study to benchmark the effects on human-exoskeleton interaction of three different exoskeleton controllers, including a new EMG-based controller designed by us and never tested in previous studies, which has made it possible to provide valuable third-party feedback on the use of the EUROBENCH facility and testbed, enriching the apprenticeship of the project consortium and contributing to the scientific community.

Plant apocarotenoids: from retrograde signaling to interspecific communication.

Carotenoids are isoprenoid compounds synthesized by all photosynthetic and some non-photosynthetic organisms. They are essential for photosynthesis and contribute to many other aspects of a plant's life. The oxidative breakdown of carotenoids gives rise to the formation of a diverse family of essential metabolites called apocarotenoids. This metabolic process either takes place spontaneously through reactive oxygen species or is catalyzed by enzymes generally belonging to the CAROTENOID CLEAVAGE DIOXYGENASE family. Apocarotenoids include the phytohormones abscisic acid and strigolactones (SLs), signaling molecules and growth regulators. Abscisic acid and SLs are vital in regulating plant growth, development and stress response. SLs are also an essential component in plants' rhizospheric communication with symbionts and parasites. Other apocarotenoid small molecules, such as blumenols, mycorradicins, zaxinone, anchorene, ß-cyclocitral, ß-cyclogeranic acid, ß-ionone and loliolide, are involved in plant growth and development, and/or contribute to different processes, including arbuscular mycorrhiza symbiosis, abiotic stress response, plant-plant and plant-herbivore interactions and plastid retrograde signaling. There are also indications for the presence of structurally unidentified linear cis-carotene-derived apocarotenoids, which are presumed to modulate plastid biogenesis and leaf morphology, among other developmental processes. Here, we provide an overview on the biology of old, recently discovered and supposed plant apocarotenoid signaling molecules, describing their biosynthesis, developmental and physiological functions, and role as a messenger in plant communication.

A manipulation of carotenoid metabolism influence biomass partitioning and fitness in tomato.

Improving yield, nutritional value and tolerance to abiotic stress are major targets of current breeding and biotechnological approaches that aim at increasing crop production and ensuring food security. Metabolic engineering of carotenoids, the precursor of vitamin-A and plant hormones that regulate plant growth and response to adverse growth conditions, has been mainly focusing on provitamin A biofortification or the production of high-value carotenoids. Here, we show that the introduction of a single gene of the carotenoid biosynthetic pathway in different tomato cultivars induced profound metabolic alterations in carotenoid, apocarotenoid and phytohormones pathways. Alterations in isoprenoid- (abscisic acid, gibberellins, cytokinins) and non-isoprenoid (auxin and jasmonic acid) derived hormones together with enhanced xanthophyll content influenced biomass partitioning and abiotic stress tolerance (high light, salt, and drought), and it caused an up to 77% fruit yield increase and enhanced fruit's provitamin A content. In addition, metabolic and hormonal changes led to accumulation of key primary metabolites (e.g. osmoprotectants and antiaging agents) contributing with enhanced abiotic stress tolerance and fruit shelf life. Our findings pave the way for developing a new generation of crops that combine high productivity and increased nutritional value with the capability to cope with climate change-related environmental challenges.

A unilateral robotic knee exoskeleton to assess the role of natural gait assistance in hemiparetic patients.

BACKGROUND: Hemiparetic gait is characterized by strong asymmetries that can severely affect the quality of life of stroke survivors. This type of asymmetry is due to motor deficits in the paretic leg and the resulting compensations in the nonparetic limb. In this study, we aimed to evaluate the effect of actively promoting gait symmetry in hemiparetic patients by assessing the behavior of both paretic and nonparetic lower limbs. This paper introduces the design and validation of the REFLEX prototype, a unilateral active knee-ankle-foot orthosis designed and developed to naturally assist the paretic limbs of hemiparetic patients during gait. METHODS: REFLEX uses an adaptive frequency oscillator to estimate the continuous gait phase of the nonparetic limb. Based on this estimation, the device synchronically assists the paretic leg following two different control strategies: (1) replicating the movement of the nonparetic leg or (2) inducing a healthy gait pattern for the paretic leg. Technical validation of the system was implemented on three healthy subjects, while the effect of the generated assistance was assessed in three stroke patients. The effects of this assistance were evaluated in terms of interlimb symmetry with respect to spatiotemporal gait parameters such as step length or time, as well as the similarity between the joint's motion in both legs. RESULTS: Preliminary results proved the feasibility of the REFLEX prototype to assist gait by reinforcing symmetry. They also pointed out that the assistance of the paretic leg resulted in a decrease in the compensatory strategies developed by the nonparetic limb to achieve a functional gait. Notably, better results were attained when the assistance was provided according to a standard healthy pattern, which initially might suppose a lower symmetry but enabled a healthier evolution of the motion of the nonparetic limb. CONCLUSIONS: This work presents the preliminary validation of the REFLEX prototype, a unilateral knee exoskeleton for gait assistance in hemiparetic patients. The experimental results indicate that assisting the paretic leg of a hemiparetic patient based on the movement of their nonparetic leg is a valuable strategy for reducing the compensatory mechanisms developed by the nonparetic limb.

Characterization and Evaluation of Human-Exoskeleton Interaction Dynamics: A Review.

Exoskeletons and exosuits have witnessed unprecedented growth in recent years, especially in the medical and industrial sectors. In order to be successfully integrated into the current society, these devices must comply with several commercialization rules and safety standards. Due to their intrinsic coupling with human limbs, one of the main challenges is to test and prove the quality of physical interaction with humans. However, the study of physical human-exoskeleton interactions (pHEI) has been poorly addressed in the literature. Understanding and identifying the technological ways to assess pHEI is necessary for the future acceptance and large-scale use of these devices. The harmonization of these evaluation processes represents a key factor in building a still missing accepted framework to inform human-device contact safety. In this review, we identify, analyze, and discuss the metrics, testing procedures, and measurement devices used to assess pHEI in the last ten years. Furthermore, we discuss the role of pHEI in safety contact evaluation. We found a very heterogeneous panorama in terms of sensors and testing methods, which are still far from considering realistic conditions and use-cases. We identified the main gaps and drawbacks of current approaches, pointing towards a number of promising research directions. This review aspires to help the wearable robotics community find agreements on interaction quality and safety assessment testing procedures.

COVID-19 Outcome Prediction by Integrating Clinical and Metabolic Data using Machine Learning Algorithms.

Background: The coronavirus disease (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus and is responsible for nearly 6 million deaths worldwide in the past 2 years. Machine learning (ML) models could help physicians in identifying high-risk individuals. Objectives: To study the use of ML models for COVID-19 prediction outcomes using clinical data and a combination of clinical and metabolic data, measured in a metabolomics facility from a public university. Methods: A total of 154 patients were included in the study. "Basic profile" was considered with clinical and demographic variables (33 variables), whereas in the "extended profile," metabolomic and immunological variables were also considered (156 characteristics). A selection of features was carried out for each of the profiles with a genetic algorithm (GA) and random forest models were trained and tested to predict each of the stages of COVID-19. Results: The model based on extended profile was more useful in early stages of the disease. Models based on clinical data were preferred for predicting severe and critical illness and death. ML detected trimethylamine N-oxide, lipid mediators, and neutrophil/lymphocyte ratio as important variables. Conclusions: ML and GAs provided adequate models to predict COVID-19 outcomes in patients with different severity grades.

Expression of a carotenogenic gene allows faster biomass production by redesigning plant architecture and improving photosynthetic efficiency in tobacco.

Because carotenoids act as accessory pigments in photosynthesis, play a key photoprotective role and are of major nutritional importance, carotenogenesis has been a target for crop improvement. Although carotenoids are important precursors of phytohormones, previous genetic manipulations reported little if any effects on biomass production and plant development, but resulted in specific modifications in carotenoid content. Unexpectedly, the expression of the carrot lycopene ß-cyclase (DcLCYB1) in Nicotiana tabacum cv. Xanthi not only resulted in increased carotenoid accumulation, but also in altered plant architecture characterized by longer internodes, faster plant growth, early flowering and increased biomass. Here, we have challenged these transformants with a range of growth conditions to determine the robustness of their phenotype and analyze the underlying mechanisms. Transgenic DcLCYB1 lines showed increased transcript levels of key genes involved in carotenoid, chlorophyll, gibberellin (GA) and abscisic acid (ABA) biosynthesis, but also in photosynthesis-related genes. Accordingly, their carotenoid, chlorophyll, ABA and GA contents were increased. Hormone application and inhibitor experiments confirmed the key role of altered GA/ABA contents in the growth phenotype. Because the longer internodes reduce shading of mature leaves, induction of leaf senescence was delayed, and mature leaves maintained a high photosynthetic capacity. This increased total plant assimilation, as reflected in higher plant yields under both fully controlled constant and fluctuating light, and in non-controlled conditions. Furthermore, our data are a warning that engineering of isoprenoid metabolism can cause complex changes in phytohormone homeostasis and therefore plant development, which have not been sufficiently considered in previous studies.

Effect of posture and body weight loading on spinal posterior root reflex responses.

The posterior root muscle response (PRM) is a monosynaptic reflex that is evoked by single pulse transcutaneous spinal cord stimulation (tSCS). The main aim of this work was to analyse how body weight loading influences PRM reflex threshold measured from several lower limb muscles in healthy participants. PRM reflex responses were evoked with 1-ms rectangular monophasic pulses applied at an interval of 6 s via a self-adhesive electrode (9 × 5 cm) at the T11-T12 vertebral level. Surface electromyographic activity of lower limb muscles was recorded during four different conditions, one in decubitus supine (DS) and the other three involving standing at 100%, 50%, and 0% body weight loading (BW). PRM threshold intensity, peak-to-peak amplitude, and latency for each muscle were analysed in different conditions study. PRM reflex threshold increased with body weight unloading compared with DS, and the largest change was observed between DS and 0% BW for the proximal muscles and between DS and 50% BW for distal muscles. Peak-to-peak amplitude analysis showed only a significant mean decrease of 34.6% (SD 10.4, p = 0.028) in TA and 53.6% (SD 15.1, p = 0.019) in GM muscles between DS and 50% BW. No significant differences were observed for PRM latency. This study has shown that sensorimotor networks can be activated with tSCS in various conditions of body weight unloading. Higher stimulus intensities are necessary to evoke reflex response during standing at 50% body weight loading. These results have practical implications for gait rehabilitation training programmes that include body weight support.

Lycopene ß-cyclase expression influences plant physiology, development, and metabolism in tobacco plants.

Carotenoids are important isoprenoids produced in the plastids of photosynthetic organisms that play key roles in photoprotection and antioxidative processes. ß-Carotene is generated from lycopene by lycopene ß-cyclase (LCYB). Previously, we demonstrated that the introduction of the Daucus carota (carrot) DcLCYB1 gene into tobacco (cv. Xanthi) resulted in increased levels of abscisic acid (ABA) and especially gibberellins (GAs), resulting in increased plant yield. In order to understand this phenomenon prior to exporting this genetic strategy to crops, we generated tobacco (Nicotiana tabacum cv. Petit Havana) mutants that exhibited a wide range of LCYB expression. Transplastomic plants expressing DcLCYB1 at high levels showed a wild-type-like growth, even though their pigment content was increased and their leaf GA1 content was reduced. RNA interference (RNAi) NtLCYB lines showed different reductions in NtLCYB transcript abundance, correlating with reduced pigment content and plant variegation. Photosynthesis (leaf absorptance, Fv/Fm, and light-saturated capacity of linear electron transport) and plant growth were impaired. Remarkably, drastic changes in phytohormone content also occurred in the RNAi lines. However, external application of phytohormones was not sufficient to rescue these phenotypes, suggesting that altered photosynthetic efficiency might be another important factor explaining their reduced biomass. These results show that LCYB expression influences plant biomass by different mechanisms and suggests thresholds for LCYB expression levels that might be beneficial or detrimental for plant growth.

Transcranial direct current stimulation combined with robotic therapy for upper and lower limb function after stroke: a systematic review and meta-analysis of randomized control trials.

BACKGROUND: Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation method able to modulate neuronal activity after stroke. The aim of this systematic review was to determine if tDCS combined with robotic therapy (RT) improves limb function after stroke when compared to RT alone. METHODS: A search for randomized controlled trials (RCTs) published prior to July 15, 2021 was performed. The main outcome was function assessed with the Fugl-Meyer motor assessment for upper extremities (FM/ue) and 10-m walking test (10MWT) for the lower limbs. As secondary outcomes, strength was assessed with the Motricity Index (MI) or Medical Research Council scale (MRC), spasticity with the modified Ashworth scale (MAS), functional independence with the Barthel Index (BI), and kinematic parameters. RESULTS: Ten studies were included for analysis (n = 368 enrolled participants). The results showed a non-significant effect for tDCS combined with RT to improve upper limb function [standardized mean difference (SMD) = - 0.12; 95% confidence interval (CI): - 0.35-0.11)]. However, a positive effect of the combined therapy was observed in the lower limb function (SMD = 0.48; 95% CI: - 0.15-1.12). Significant results favouring tDCS combined with RT were not found in strength (SMD = - 0.15; 95% CI: - 0.4-0.1), spasticity [mean difference (MD) = - 0.15; 95% CI: - 0.8-0.5)], functional independence (MD = 2.5; 95% CI: - 1.9-6.9) or velocity of movement (SMD = 0.06; 95% CI: - 0.3-0.5) with a "moderate" or "low" recommendation level according to the GRADE guidelines. CONCLUSIONS: Current findings suggest that tDCS combined with RT does not improve upper limb function, strength, spasticity, functional independence or velocity of movement after stroke. However, tDCS may enhance the effects of RT alone for lower limb function. tDCS parameters and the stage or type of stroke injury could be crucial factors that determine the effectiveness of this therapy.